The overall goal of this project is the development of new adenovirus- based gene transfer vectors that are better suited than those that are currently available for human gene transfer therapy of cystic fibrosis and other genetic diseases. The specific aims of this development include: 1) helper-dependent adenoviral vectors that accommodate a high molecular weight DNA insert; 2) site-specific chromosomal integration (safe harbor) by legitimate recombination; 3) long-term tissue-specific expression; and 4) control of expression by small molecular weight inducers (drugs). These objectives, if achieved, will overcome three major disadvantages of the adenovirus system: lack of efficient nuclear integration, limited insert packaging capacity, and control of expression in particular cell types. An adenovirus amplicon plasmid with a minimum cis-element for packaging of adenovirus genome will be constructed. This vector has the potential for creating an adenovirus-based vector with capacity of an insert of up to 37 kb. Such a vector will remove size limitations for inclusion of cis- regulatory elements and will also open a new possibility to add a feature of efficient nuclear targeting by homologous recombination. The gene targeting efficiency of adenoviral vectors will be studied in mouse and human chromosomal sites. Attempts to target in vivo will be performed in mice and baboons. We propose to introduce a drug-responsive promoter, Cypla-1, into adenoviral vectors and examine the inducibility in vivo. If successful, these advances could lead to a long-term expression capable of drug-mediated regulation.